Systematic Review of Newborn Screening Programmes for Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder causing the degeneration of motor neurons in the spinal cord. Recent studies suggest greater effectiveness of treatment in the presymptomatic stage. This systematic review synthesises findings from 37 studies (and 3 overviews) of newborn screening for SMA published up to November 2023 across 17 countries to understand the methodologies used; test accuracy performance; and timing, logistics and feasibility of screening. All studies screened for the homozygous deletion of SMN1 exon 7. Most (28 studies) used RT-PCR as the initial test on dried blood spots (DBSs), while nine studies also reported second-tier tests on DBSs for screen-positive cases. Babies testing positive on DBSs were referred for confirmatory testing via a range of methods. Observed SMA birth prevalence ranged from 1 in 4000 to 1 in 20,000. Most studies reported no false-negative or false-positive cases (therefore had a sensitivity and specificity of 100%). Five studies reported either one or two false-negative cases each (total of six cases; three compound heterozygotes and three due to system errors), although some false-negatives may have been missed due to lack of follow-up of negative results. Eleven studies reported false-positive cases, some being heterozygous carriers or potentially related to heparin use. Time to testing and treatment varied between studies. In conclusion, several countries have implemented newborn screening for SMA in the last 5 years using a variety of methods. Implementation considerations include processes for timely initial and confirmatory testing, partnerships between screening and neuromuscular centres, and timely treatment initiation.


Introduction
Spinal muscular atrophy (SMA) is an autosomal recessive disease associated with the progressive and irreversible degeneration of lower motor neurons in the anterior horn of the spinal cord and brainstem.The onset of neuromuscular weakness ranges from birth to adulthood.Historically, SMA was classified into discrete types based on age of onset of weakness, with SMA type 0 presenting neonatally and type 4 in early adulthood.It is now apparent that SMA spans a continuum without discrete subtypes.The vast majority of cases of SMA (95%) are due to a homozygous deletion of exons 7 and 8 of SMN1 [1].A minority are compound heterozygotes, where one copy of SMN1 is deleted and the other has a missense variant.Overall, these genetic changes lead to a decrease in functional SMN protein and ultimately lead to patients developing SMA.The related SMN2 gene can also make SMN protein, but only around 10% of the SMN protein from the SMN2 gene is functional.Therefore, SMN2 can partially compensate for deletions or mutations in SMN1.People can have multiple copies of the SMN2 gene with a higher number of SMN2 copies generally correlating with reduced disease severity [2].However, it is not currently possible to accurately predict severity from genetic information alone.
Many countries have begun to introduce newborn screening for SMA.Newborn screening aims to identify babies with SMA via the screening of all newborns in a country or area.Newborn screening for SMA often uses real-time quantitative polymerase chain reaction (qRT-PCR) techniques to assess the patient's SMN genes, using DNA isolated from dried blood spots (DBSs) collected soon after birth.Most newborn screening for SMA screens for homozygous deletion of the SMN1 gene.
Treatments for SMA include nusinersen (Spinraza) [3], an antisense oligonucleotide designed to modify the product of the SMN2 gene to produce more functional SMN protein, risdiplam (Evrysdi), a small molecule drug that targets the SMN2 gene to produce more SMN protein [4], and onasemnogene abeparvovec (Zolgensma), a gene therapy which expresses the SMN protein [5].Recently, treatment of SMA in the presymptomatic stage has been suggested to improve outcomes compared to the treatment of symptomatic disease [6].Presymptomatic treatment may be facilitated by identifying babies at an early stage via newborn screening [7].
We therefore undertook a systematic review of cohort studies of newborn screening for SMA worldwide to understand the methodologies used and the ability of screening to reliably identify neonates with SMA in the presymptomatic stage.

Aims of Review
This systematic review aimed to synthesise findings from cohort studies of newborn screening for SMA worldwide to understand the methodologies used; the numbers and potential causes of false-negative and false-positive cases; the test accuracy of screening; and findings relating to the timing, logistics and feasibility of screening.Our systematic review followed the PRISMA guidelines.Our review protocol was registered on PROSPERO (registration number CRD42023473172).

Search Strategy
Searches of MEDLINE, Embase and the Cochrane Library were conducted in November 2023 and covered all dates up to this point.Thesaurus and free-text terms for SMA (plus synonyms) were combined with terms for newborn screening.The search strategy is provided in Appendix A. Recent reviews and relevant studies were also checked, and experts consulted, to identify any additional studies.

Inclusion and Exclusion Criteria
The review included studies of newborn screening for 5q SMA worldwide.Prospective cohort studies and RCTs were eligible for inclusion, while case-control studies were not included.However, a systematic search for case-control studies was undertaken, and a list is provided in Appendix B (Table A1) for information.Studies of both pilot and routine screening were eligible.Relevant outcomes included the observed birth prevalence of SMA; numbers and potential causes of false-negative and false-positive cases; test accuracy outcomes (sensitivity, specificity, positive and negative predictive value); and findings relating to the timing, logistics and feasibility of screening.This review focusses on screening processes and diagnostic follow-up, and it does not seek to evaluate ongoing patient management, patient outcomes or loss to follow-up.

Study Selection and Data Extraction
References were checked for inclusion by one reviewer, and a 10% sample was checked by a second reviewer early in the process to check for consistency in inclusion decisions.Data for all studies were extracted by one reviewer and checked by another.Data were extracted relating to the country/area, whether pilot or routine screening, dates of screening, methodologies for initial and confirmatory testing, and outcomes as listed above.

Risk of Bias Assessment
Risk of bias within included studies was assessed using the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) tool [8].

Calculation of Outcome Measures
Test accuracy outcomes were reported as stated in included studies or calculated by the review team where data permitted.As an overview of test accuracy outcomes, truepositive (TP) cases are those who test positive and truly have the condition; true-negative (TN) cases are those who test negative and truly do not have the condition; false-positive (FP) cases are those who test positive but do not have the condition; and false-negative (FN) cases are those who test negative but do actually have the condition.From these numbers, the following test accuracy outcomes were calculated.The positive predictive value is the number of patients correctly testing positive as a percentage of all those with a positive initial test result (TP/[TP+FP]).The negative predictive value is the number of patients correctly testing negative as a percentage of all those with a negative initial test result (TN/[TN+FN]).Sensitivity is the number of patients correctly testing positive as a percentage of all those who truly have the condition (TP/[TP+FN]).Specificity is the number of patients correctly testing negative as a percentage of all those who truly do not have the condition (TN/[TN+FP]).
The aim of most screening programmes was to detect homozygous deletions of SMN1.Most screening methods were not designed to identify compound heterozygotes of SMN1 (around 2-5% of SMA cases).Therefore, sensitivity was calculated in two ways: firstly for detecting homozygous SMN1 deletions and secondly for detecting any SMA case (including both homozygous deletions and compound heterozygotes; this latter measure would be expected to be a maximum of 95-98%, since compound heterozygotes would not be identified).
In addition, some studies reported conducting "second-tier" (and sometimes "thirdtier") testing on the original DBS when the initial screening result was positive or inconclusive.These additional tests on the original DBS were considered part of the index test when calculating test accuracy outcomes.Conversely, the confirmatory test on a new blood sample, generally conducted in a specialist centre, was considered the reference standard test.

Synthesis Methods
Findings were synthesised via tabulation and narrative synthesis.

Volume, Type and Setting of Included Studies
The search generated 494 references from the database search and 1 from other sources.In total, 40 studies were included (within 53 references; Table 1).A PRISMA flow diagram is shown in Figure 1.
The review identified 37 cohort studies of newborn screening for SMA .No RCTs of newborn screening were identified.Of the 37 cohort studies, 34 studies reported prospective screening programmes of newborns using DBS screening, while three studies reported analyses using cohorts of anonymised DBS samples (one in Ohio [44], two in China [54,55]).Of the 34 prospective screening studies, 22 were pilot studies, 9 were routine screening, and 3 were both.In terms of location, four studies reported nationwide screening (in Germany [17], Latvia [21], Norway [25] and Japan [50]), while 29 covered a particular area or state (and one did not report this [22]).The majority of included references were published between 2019 and 2024, reflecting the recent nature of published studies.
In addition, we identified three overviews of screening studies across broader geographical locations (one global, one USA-based and one Canada-based); these overviews reported data on prevalence, screening methodologies and diagnostic accuracy, and they were therefore includable in our review [58][59][60][61].The global overview published in 2021 suggested that by 2025, newborn screening for SMA was forecast to include 24% of newborns in countries where a disease-modifying drug is available and 8.5% of newborns in countries with no disease-modifying drugs [58].An overview for Canada reported that SMA newborn screening was available in five of eight Canadian provinces and all three territories by October 2022, and that the number of Canadian newborns screened for SMA increased from 60% in June 2022 to 72% in January 2023 [59].A similar overview for the USA reported that SMA newborn screening was available in 48 of 53 US states or territories as of December 2022 [60,61].

Prevalence of SMA from Newborn Screening Studies
The total number of newborns screened per study (across the 34 prospective studies) ranged from 2552 to 650,000 (Table 1).The number of identified SMA cases ranged from 0 to 43.Based on these data, the observed prevalence of SMA ranged from around 1 in 4000 to 1 in 20,000 (Table 1).It is possible that some SMA cases were not detected via screening, firstly because most screening programmes are not designed to identify compound heterozygotes (2-5% of SMA cases) and secondly because some false-negative cases may have been missed if they were not diagnosed clinically within the study timeframe.This could mean that prevalence is underestimated in some studies.1.All studies aimed to screen for the homozygous deletion of SMN1 exon 7 so would not identify compound heterozygotes.However, some studies also identified heterozygous carriers, including the New York State pilot study [40], a study in Norway [25], a study in Russia [57] and a study in China using anonymised DBS samples [54].In New York State [40], parents of heterozygous carriers were offered genetic testing to determine whether both parents were carriers.In the Norwegian study [25], babies with a heterozygous deletion were further tested for a specific point mutation, so compound heterozygotes with this mutation would have been identified.
SMA screening was reported to be multiplexed with screening for severe combined immunodeficiency (SCID) in around 40% of studies (15 of 37), including studies in the USA, Canada, Australia, Germany, Italy, Norway, Japan and Taiwan.In addition, a few studies reported multiplex screening with other conditions, including SCID plus sickle cell disease (Germany [18]); SCID plus B-cell deficiency (Japan [47]) or SCID plus hearing loss (Canada [30]).Table 1 also notes whether programmes used their own lab-developed test or a commercial test; this varied between studies but was often unclear from the study report.
In all studies, screen-negative cases were not followed up further.Screen-positive cases could undergo three types of further testing, as described below: (i) second-tier testing for SMN1 deletion on the original DBS; (ii) referral to a specialist centre for confirmatory testing of SMN1 deletion on a fresh blood sample; and (iii) testing for SMN2 copy number.

Methodologies for Second-Tier Testing of DBS Sample
Here, we refer to "second-tier testing" as any further testing for SMN1 deletion on the original DBS for screen-positive cases.Some but not all studies included second-tier tests.In total, 12 studies reported repeating the initial PCR on screen-positive cases.Furthermore, nine studies conducted other types of second-tier test on the original DBS for screen-positive cases, including droplet digital PCR (ddPCR, n = 3) [33,42,51], multiplex ligation-dependent probe amplification (MLPA; n = 3) [10,23,30], restriction fragment length polymorphism PCR (RFLP-PCR; n = 3) [23,50,56], and one study with three-tier testing in screen positives (Massachusetts: PCR, then testing for exon 7 variants, then sequencing [36]).These secondtier tests on the DBS were considered part of the index test rather than the reference standard within this review when determining test accuracy.

Overview of Test Accuracy Data
Most cohort studies reported the total number of newborns screened, the number testing positive, and the number of true-positive and false-positive cases.Confirmatory testing on a new blood sample was only performed on babies who tested positive in the initial screen.Therefore, false-negative cases (those missed by screening) were generally only identified if they later presented with symptoms, and so numbers of false-negative cases may have been underestimated, particularly later-onset cases of SMA which may not be clinically apparent in early life.Some studies did not mention false-negative cases at all, so it was unclear whether information on missed cases had actually been sought.
The numbers of false-positive and false-negative cases, and associated test accuracy outcomes, are summarised in Table 2.

Positive Predictive Value
It was generally possible to calculate the positive predictive value; however, this was based on small numbers of cases.Due to the low prevalence, a small number of falsepositives could substantially reduce the positive predictive value.Where this could be calculated, it was 100% in 15 studies [10,13,18,19,21,30,33,38,40,42,44,46,53,56,57], and in the remainder, it was 4% [55], 17% [49], 38% [34], 50% [41], 69% [35], 80% [45], 83% [32], 90% [36], 92% [17], 93% [43] and 95% [26].A lower positive predictive value means that a study had more false-positives.As noted earlier, second-tier and third-tier tests on the original DBS were considered part of the index test when calculating test accuracy outcomes, while confirmatory testing on a new blood sample in a specialist centre was considered the reference standard.If only the first-tier test was considered to be the index test, the positive predictive value would be lower, as some false-positives are ruled out during subsequent tiers of testing on the DBS.Possible reasons for false-positives are discussed below and summarised in Table 3.

Negative Predictive Value
The negative predictive value could generally be calculated, but it may be overestimated due to the underestimation of false-negative cases, as described above.Where the negative predictive value could be calculated, it was 100% in all studies (to the nearest whole percentage point).This was the case even where a study reported some false-negatives due to the low prevalence of SMA in the population.

Sensitivity
It was generally possible to calculate sensitivity, but again, this may be overestimated due to the underestimation of false-negative cases.Also, due to the low prevalence, a small number of false-negatives could substantially reduce the sensitivity.As noted in the Methods, sensitivity was calculated in two ways: firstly for detecting homozygous SMN1 deletions (which were the target of screening), and secondly for detecting any SMA case (including compound heterozygotes which could not be identified via screening).Sensitivity for detecting homozygous SMN1 deletions (where calculable) was 100% in 23 studies, and it was 91% and 94% in two further studies with two and one false-negative cases, respectively [26,34].In addition, three studies each identified one compound heterozygous case (identified via symptoms and classed as false-negative); the sensitivity for these studies, calculated for all SMA cases rather than just homozygous deletions, was 90%, 95% and 98% [10,17,51].

Specificity
Specificity could generally be calculated, because the number of false-positive cases was generally reported.Where specificity could be calculated, it was 100% in all studies (to the nearest whole percentage point).This was the case even where a study reported some false-positives due to the low prevalence of SMA in the population.

False-Negatives, False-Positives, Incomplete Results and Incidental Findings
Details and possible causes of false-positive and false-negative cases, as well as initial incomplete results and incidental findings, are provided in Table 3.

False-Negative Cases
The majority of studies did not report any false-negative cases.Only six false-negative cases were reported across five studies [10,17,26,34,52]; these babies were generally identified when they presented with symptoms.Three false-negative babies were found to be compound heterozygotes, which cannot be identified via screening for homozygous deletions of SMN1 [10,17,52].Three further false-negative cases were related to system or human errors [26,34] (Table 3).

False-Positive Cases
The majority of studies (eighteen studies) did not report any false-positive cases, while six studies reported one false-positive each [26,32,36,41,43,45], and one study each reported 4 false-positives [17], 5 false-positives [35], 10 false-positives [49], 22 false-positives [55] or 24 false-positives [34] (Table 3; the remaining studies did not report this information).False-positives were identified upon confirmatory testing on a new blood sample.Some false-positives were found to be heterozygous carriers of the SMN1 deletion [17,32,45], or had sequence variants in the SMN1 or SMN2 genes [26], or recombination between the genes [52].Some babies with false-positive results were unwell in hospital at the time of sample collection [34], or premature [34], or also had a false-positive SCID screen [35]; the correlation between these factors and a false-positive result was unclear.Some falsepositive cases were suggested to be due to heparinised and/or diluted blood in the DBS sample [49] (Table 3).

Initial Incomplete Results
Thirteen studies reported cases with incomplete or uncertain results on the initial test, who then had a definitive result on further tiers of testing [19,21,[32][33][34]36,40,41,44,45,47,52,56] (these were not classed as false-positives since the issues were resolved through further testing of the initial DBS sample, which was considered to be part of the index test process).Some were thought to be due to the use of heparin [19]; some related to babies in the neonatal intensive care unit (NICU), possibly due to presence of a PCR inhibitor [36]; some were due to poor DNA quality or quantity [21,33,40,41,44,45,47,52]; some were due to system or handling errors [32]; and some were not explained further (Table 3).

Incidental Findings, Sibling Diagnosis and Sequence Variants
Four studies reported cases of siblings being diagnosed with SMA following a positive screening case [10,13,19,56], and one study reported the identification of an unrelated blood disorder [41], while two studies reported initial uncertain results relating to variants of uncertain significance in SMN1 exon 7 [36,40] (further details in Table 3).

Risk of Bias in Included Studies
Risk of bias in the included studies is shown in Table 4.The included studies were assessed using the QUADAS-2 quality assessment tool, which was tailored to the review question.
In terms of patient selection, 37 of 40 studies were considered to have a low risk of bias due to being cohort studies including a consecutive or random sample of patients (Table 3).Regarding the index test, 39 of 40 studies were considered to have a low risk of bias since the index tests were interpreted without knowledge of the reference standard and did not require the consideration of different thresholds.Furthermore, all the included studies had low concern for applicability for patient selection, index test and reference standard domains, apart from one study [54] being unclear in the reference standard domain.
However, all studies (n = 40) were considered to have a high risk of bias for the "reference standard" and "flow and timing" domains, because screen-negative patients did not undergo confirmatory testing, and the results of the index test were likely to have been known when interpreting the reference standard.

Timing of Testing Process
Some studies noted timings of the testing process; timings from birth are reported in Table 5. Median time from birth to DBS sampling was generally 1-6 days, and median time from birth to DBS receipt at the screening centre was generally 2-6 days (or 75 days in one study).Median time from birth to initial screening results ranged from 3 to 18 days.Median time from birth to specialist consultation ranged from 5 to 33 days, while confirmatory results on a new blood sample were available at a median age of 11-28 days.Treatment start was more variable, as it was reported as occurring at a median age of 15-48 days (or 106 days in one study).
Some studies reported the point at which parents were contacted.This was often on the same day as, or soon after, the positive screening result with a specialist appointment arranged for soon after this for examination and confirmatory blood test.

Workflow and Consent Processes
Table 6 summarises information on workflow and consent processes.In terms of workflow, studies varied widely in terms of volume of samples processed, which ranged from 300 per week to 2000 per day.Some screening programmes used opt-in processes and some used opt-out processes.Where reported, consent rates were generally high (over 90%), and this increased when SMA became part of routine screening.

Organisational Considerations, Implementation and Barriers
Some studies reported on organisational and implementation issues and barriers or delays to treatment, as summarised in Table 7.

Start of Treatment
Table 6.Workflow and consent.

Study, Location Consent processes
Global overview [58] Some countries use opt-in (Germany, Italy, Japan, Taiwan, Russia) and some opt-out (USA, Canada, Belgium, Australia) Canada overview [59] Most provinces screen for SMA alongside other newborn screening and do not require specific consent, while Alberta has an opt-out process UK (Thames Valley) [9] Initial uptake of antenatal consent was slow with staff availability the main limiting factor.Consent rate increased with remote consenting and with postnatal consent during baby checks Table 6.Cont.

Study, Location Implementation and Barriers
Global overview [58] Implementation considerations:

•
Changing access to presymptomatic disease-modifying therapies; also limited access for 3+ SMN2 copies Canada overview [59] Barriers: • Most Canadian provinces require a positive confirmatory genetic test prior to application for treatment, which can result in an additional 1-2 week delay in initiating treatment, while Saskatchewan allows application after a positive initial screen

Study, Location Implementation and Barriers
Canada overview [59] Implementation considerations: Modifications that could potentially reduce time to treatment initiation:

•
For 2 cases, clinicians noted delays may have been compounded by the new process for newborn screening USA (Kentucky) [35] Barriers: Factors causing delayed treatment: USA (New York State) [38,39] Barriers: • Medical delays most commonly reported were the presence of AAV9 antibodies and elevated troponin I levels • Nonmedical barriers included delays in obtaining insurance and insurance policies regarding specific treatment modalities Japan (Osaka) [47,48] Barriers: • Some samples were delayed in arriving at the lab, mainly due to problems with transportation over weekends and public holidays Russia (Moscow) [56] Barriers: Logistical issues: The most commonly cited barriers leading to delayed treatment were related to (a) testing, e.g., requirement to obtain confirmatory testing results prior to application for treatment; (b) medical issues, e.g., SMA-related or other health issues; (c) financial issues, e.g., problems with insurance authorisation or reimbursement of treatment; and (d) logistical issues, e.g., delayed arrival of the samples at the lab due to problems with transportation, and transporting patients to the centre for confirmatory testing and treatment.
Included studies highlighted some points to be considered before SMA newborn screening is implemented as routine screening at the national or regional level.These included (a) beginning with a pilot project; (b) establishing a well-thought-out implementation process, including developing the screening assay, staffing, selection of specialist centres, funding, regulatory requirements, and process for follow-up care and presymptomatic treatment; (c) logistical considerations, e.g., operation of screening laboratories on weekends, reduction in time to transport samples from the collection site to screening laboratories, and time required for confirmatory testing and treatment approval; and (d) establishing partnerships between newborn screening staff and neuromuscular specialists and patient organisations to reduce delays and promote family-centred care.
Additional ongoing uncertainties included treatment cost-effectiveness and reimbursement; uncertainty regarding long-term outcomes for presymptomatic patients; and uncertainties about management of patients with ≥4 SMN2 copies.

Discussion
This review identified 34 prospective cohort studies (plus three overviews and three cohort analyses of anonymised DBSs) evaluating pilot or routine newborn screening for SMA across 17 countries.All studies screened for homozygous deletion of SMN1 exon 7. Most (28 of 37) used RT-PCR to detect homozygous SMN1 deletion, and nine studies included additional second-tier tests on dried blood spots (DBSs) for screen-positive cases.Around 40% multiplexed SMA screening with screening for severe combined immunodeficiency (SCID).Babies testing positive via DBSs were referred for confirmatory testing on a new blood sample via MLPA, RT-PCR, ddPCR, RFLP-PCR or sequencing.
Across studies, six false-negative cases were identified via symptoms: three compound heterozygotes and three due to system errors.False-positive cases ranged from n = 0 to n > 10; some were heterozygous carriers or potentially related to heparin use.The positive predictive value ranged from 4% to 100% depending on the false-positive rate.Sensitivity was 100% in most studies, although some false-negatives may have been missed.The specificity and negative predictive value were close to 100% due to the low prevalence of SMA.Time to testing and treatment varied between studies.
The identification of false-positive cases and initial incomplete results (for example due to heterozygosity for SMN1 deletion, SMN gene sequence variants, gene recombination, presence of PCR inhibitors or issues with DNA quality or quantity) highlights the importance of confirmatory testing.This may include second-tier testing on the initial DBS, which may rule out some false-positive cases without anxiety to families as well as confirmatory testing on a new blood sample.Furthermore, confirmatory testing together with genetic counselling in a clinical setting may ensure the cascade testing of family members, identify family members at risk of developing SMA, and provide information regarding family planning.
The majority of included references were published between 2019 and 2024, reflecting the fact that newborn screening is currently being piloted, evaluated or implemented in several countries worldwide.Previous reviews of newborn screening for SMA [62][63][64][65][66][67] have generally identified smaller numbers of studies due to the volume of articles reported very recently.
Observed prevalence estimates for 5q SMA ranged from 1 in 4000 to 1 in 20,000, which tallies with the reported prevalence of 1 in 6000 to 1 in 30,000 in a recent review [68].The apparently wide variation in estimates may be due to the small numbers of cases identified in the various studies (so, for example, one missed case may change the estimate).
In terms of limitations, some information was not well reported, such as the reasons for inconclusive or false-positive results.The test methods for the various tiers of DBS testing, confirmatory testing, and SMN2 copy number testing were not always clearly reported, and the review indicates that there is still relatively wide variation in the methods used.
Further research may focus on the most appropriate testing methods for both DBSs and confirmatory testing as well as the potential for adding SMA screening into routine newborn screening processes.Further work on implementation factors may inform how best to facilitate the timely identification and treatment of patients at a presymptomatic or early symptomatic stage.Our review does not seek to evaluate ongoing patient management, patient outcomes or loss to follow-up of screened babies, but such information would be valuable in order to understand whether SMA screening programmes are fulfilling their potential in enabling the early management of babies with SMA.There are also ongoing uncertainties around managing patients with four SMN2 copies who may not have been diagnosed until much later in life in the absence of screening.

Conclusions
In the last five years, several countries have evaluated newborn screening for SMA.Across 37 studies, 6 false-negative cases were identified, while false-positive cases per study ranged from 0 to more than 10.Positive predictive value ranged from 4% to 100%; sensitivity was 100% in most studies; while specificity and negative predictive value were close to 100% due to the low prevalence of SMA.Implementation considerations include processes for timely initial and confirmatory testing, partnerships between screening and neuromuscular centres, and timely treatment initiation.

Table 1 .
Methodologies of screening for SMA.

Table 2 .
Test accuracy of screening for SMA.

Table 4 .
Risk of bias in included studies.

Table 4 .
Cont.On each "risk of bias overall" criterion, studies scored Low if Y to all individual criteria, High if No to any criteria, and Unclear if some criteria were Unclear but none scored Low.

Table 5 .
Timing of testing process.